Dominance-associated traits in male mammals such as aggression and territoriality have long been viewed as ecologically significant. It has been found that the dominant male mouse tends to sire more litters than the subordinate male mouse. Subordinate male mice occupy less desirable habitats, have a lower survival rate, and a lower reproductive potential than dominant male mice. In addition, there are numerous physiological attributes of subordination, such as poor spermatogenesis, decreased gonadal activity and abnormal adrenal function. The reproductive success of the dominant males has been previously and only partially explained by the fact that dominant males are known to deter subordinates from approaching "receptive" female mice. The dominant male produces an "aversion signal" that drives the subordinate male mouse away. This aversion signal is purportedly produced by secretions of the preputial gland of male mice. The preputial gland is believed to be a source of androgen-dependent sex-related pheromone. It is therefore desirable to utilize as mouse population controls, those chemical compounds which are responsible for the aversive signal since such compounds are capable of inhibiting the sexual activity of a male mouse and controlling the population growth of mice.
Mouse urine has been shown to be a rich source of olfactory cues which elicit changes in the reproductive behavior and physiology of recipient animals. The male urinary pheromones induce the pregnancy block, estrous synchronization, puberty acceleration, sexual attraction and interamale aggression and establish territory and social ranking. These pheromones are androgen-dependent and are not present in the urine of castrated mice. Social subordination is associated with suppressed gonadal function in male laboratory mice and brown lemmings, inhibited scent marking, suppressed ability of male urine to accelerate the onset of puberty in females and decreased ability of the urine to cause pregnancy blockage.
While it has long been assumed that urinary constituents, in a qualitative or quantitative sense, may signal the dominance of male mice, no chemical data had yet been reported on this subject prior to the present invention. However, the inventors have previously isolated urinary components under the control of the adrenal gland of the female mouse which have been effective in delaying the onset of puberty in female mice. These components are n-pentyl acetate, cis-2-penten-1-yl acetate and 2,5-dimethylpyrazine. levels of testosterone over subordinates and the dominant mouse's urine has an odor indicative of social status, the difference in the urinary volatile profiles of trained fighter males and subordinate males was then investigated. Several different pheromones were thoroughly investigated.
The first mouse pheromone effect to be thoroughly described by the present inventors was the promotion of inter-male aggression by dehydro-exo-brevicomin and 2-(sec-butyl)-4,5-dihydrothiazole. After detailed studies of the behavioral activity of these two compounds, it was discovered that they elicit vigorous and persistent antagonistic behavior from castrated male mice. The response provided by these two compounds is statistically indistinguishable from that found in normal male mice. The fact that these compounds are not active when spiked into water suggests that other compounds may be involved. Such additional compounds may simply provide a familiar and meaningful context in which to perceive the pheromones. 2-sec-butyl-4,5-dihydrothiazole and 3,4-dehydro-exo-brevicomin also account for at least two more pheromone effects. These compounds act synergistically to attract female mice when presented in the context of castrated male urine. It was also found that these two compounds were capable of increasing the frequency of estrus in females which were caged in high-density population conditions. For this pheromone effect, the two compounds spiked into water were sufficient to elicit a response. The effect of the synthetic compounds was similar to that of normal male urine, except that the response was somewhat attenuated when tested on females living under low-density population conditions.
The majority of work done on the preputial gland has involved the chemical characterization of lipids produced by this gland and their ramifications as related to dermatology. The preputial gland of the mouse is a specialized, testosterone-dependent sebaceous gland which is easily isolated as a pure preparation. Since obtaining a pure preparation of sebaceous glands from skin is difficult, preputial glands have been used as a model system for investigating the biochemistry of sebaceous glands. The primary orientation of past investigations have not emphasized the chemical signaling (i.e., "aversive signal") function of this gland.
Studies reveal that the chemical composition of preputial gland lipids depends on the age and sex of mice. Analysis of glands from immature male mice indicated that sterol esters and triglycerides were the principal lipids present. Acetates account for 5% of lipids in the preputial glands from male mice and 1% of the lipids in glands from female mice. Further investigation and chemical characterization of the semivolatile components of the preputial gland is described in the present invention.
Two sesquiterpenic constituents of the preputial gland of the mouse have been isolated and shown to be responsible for the "aversive signal" resulting in significantly discouraging prolonged territorial and sexual investigation by male mice, according to the present invention. These two constituents, which are the territorial markers used by dominant male mice, have been identified as E,E-alpha-farnesene and E-beta-farnesene.
Previously, alpha- and beta-farnesene have been shown to be present as naturally occuring compounds in various plant materials. The farnesyl pyrophosphate is a well-known key intermediate in the biosynthesis of steroids. Alpha-farnesene was isolated by Murray (Aust J. Chem., 22: 197, 1969) from the natural coating of apples. Spectroscopic studies verified the trans configuration of the double bond at the 3,4 position but the author was unable to verify the geometry at the 6,7 position. Later studies by Anet (Aust. J. Chem., 23: 2101, 1970) showed the apple farnesene to be the E,E-alpha-farnesene. Alpha-farnesene has also been isolated from pears and quinces. Z,E-alpha-farnesene has been isolated from the oil of Perrila frutscens f. viridis, an Asiatic mint, by Sakai and Hirose, Bull. Chem. Japan, 42: 3615, 1969. These authors also isolated the cis and trans allofarnesenes from the sesquiterpene fraction of this oil. Analysis of the essential oil of hops revealed the presence of both alpha- and beta-farnesene. As already indicated, trans-beta farnesene has also been isolated from the oil of chamomile.
The farnesenes have been isolated from several animal sources. The springbok releases an exudate from its dorsal gland which is thought to function as a conspecific alarm signal. Among the compounds found in this secretion are alpha- and beta-farnesenes. Male Mediterranean fruit flies release a volatile pheromone which attracts and excites female flies. Baker, et al. (J. Chem. Soc., Chem. Commun., 12: 842, 1985) identified nine compounds from the anal ampoule of male flies, of which E,E-alpha-farnesene is the major component.
A large body of evidence illustrates that the farnesenes are utilized by certain insects as pheromones. Ants produce a trail pheromone which is secreted by Dufour's gland. Initial studies showed that this gland's secretion was homogeneous and identical to the alpha-farnesene found in the coating of apple (see, e.g., Cavill, et al. Tetrahedron Lett., 23: 2201, 1967; Murray, supra). More detailed studies analyzing whole-worker extracts of ants isolated four fractions which exhibit trail-following activity. The major active component was identified as Z,E-alpha-farnesene. E,E-alpha-farnesene was found in lower concentrations and also showed activity. The third and fourth active fractions were tentatively identified as homosesquiterpenes having the structures of Z,Z- and Z,E-3,4,7,11-tetramethyl-1,3,6,10-dodecatetrene. Vander Meer, et al. (Tetrahedron Lett., 22: 1651, 1981) tested all six alpha- and beta-farnesene isomers and found Z,Z-alpha, E,Z-alpha-, E-beta-, and Z-beta-farnesenes inactive.
Farnesenes are also utilized as pheromones by aphids. When presented with a predator, the aphids release an alarm pheromone identified as E-beta-farnesene, from their cornicles which causes conspecifics to drop from their feeding spots, thus escaping the predator. After dropping from the plant, the aphid remains immobilized for a short period. This post-exposure quiescence is sometimes referred to as "feigning thanatosis". E-beta-farnesene shows biological activity in at least ten aphid species, making this the most interspecially active pheromone known. While studying the volatile components of Myzus persicae, Pickett and Griffiths (J Chem. Ecol., 6: 349, 1980) also found small amounts of E,E- and Z,E-alpha-farnesene. These alpha-farnesenes do not elicit an alarm response in the aphids; however, they may potentiate the effects of E-beta-farnesene.
The biological precursors for alpha- and beta-farnesene, in the preputial gland, are ocimene and myrcene. It is well established that geranyl pyrophosphate condenses with the five-carbon isopentenyl pyrophosphate to form farnesyl pyrophosphate. The farnesyl pyrophosphate is a key intermediate in steroid biosynthesis. It appears that an analogous metabolic pathway is present in the preputial gland of the male mouse; myrcene or ocimene units may condense with geranyl pyrophosphate to form E-beta-farnesene and E,E-alpha-farnesene, respectively. Absence of the farnesenes from bladder urine and identification of these compounds in the preputial glands identifies this gland as the site of secretion of these compounds. Additionally, identification of the biological precursors of the farnesenes, myrcene and ocimene, implicates this gland as the site of biosynthesis of the farnesenes. The chemical structures of myrcene, ocimene, E,E-alpha-farnesene and E-beta-farnesene are represented below. ##STR1##